The present invention relates to recombinant DNA and proteins encoded thereby having use in provision of vaccines, diagnostics test kits and methods of diagnosis for equine arteritis virus (EAV) and equine arteritis virus mediated disease.
Equine viral arteritis, a disease for which horses and donkeys are the only reported hosts, has been known for some 40 years and manifests itself with widely varying clinical signs. In its most severe form EAV infection causes abortion which makes it a potentially significant commercial threat to, inter alia, the race horse breeding industry. Early veterinary articles refer to it as epizootic cellulitus pinkeye or equine influenza. Disease outbreaks are identified infrequently and field isolates of the single stranded RNA virus itself are rare.
The virus is transmitted by the respiratory and venereal routes, with a 30% carrier state existing in seropositive stallions making the latter route a particular cause for concern as these shedding stallions may consequently infect brood-mares. In the light of the potential economic importance of the virus and its stud carrier mediated infection capability there exist a requirement for both prophylactic treatment and reliable diagnosis of EAV.
Laboratory tests based upon ELISA, virus neutralisation (VN) and complement fixation (CF) formats have been developed (see Chirnside (1992) Br. vet. J. 148 pp181). The known ELISA is relatively insensitive when applied to tissues, eg. sera, from horses previously vaccinated for other diseases such as influenza and herpesvirus, while the VN and CF formats have limited temporal sensitivity; the VN test is unable to distinguish between vaccination and natural infection.
Vaccination procedures have concentrated on safety and efficacy of whole inactivated virus and attenuated live virus vaccine. The live vaccine can induce shedding of virus from the nasopharynx and does not prevent this causing infection of commonly housed animals that have not been so treated. The known formalinised vaccine does not provide reliable protection.
Attempts to provide improvements to both diagnostic tests and vaccines have included studies into panels of antibodies raised against various EAV proteins. A 29K envelope protein in particular has been identified as antigenic and capable of causing production of neutralising antibodies in mouse (Balasuriya et al (1993) Journal of General Virology, 74, p2525-2529). The identity of this protein is unknown but work reported since the priority date of the present application by Deregt et al (J. General Virology 75, pp2439-2444) has shown that some monoclonal antibodies raised to GL protein are EAV neutralising, as are those to the nucleocapsid N protein. Results of tests in horse have yet to be reported.
The present inventor now provides isolated peptides that produce a potent neutralising immune response against EAV when administered to animals, particularly horses, and these peptides provide sensitive detection of EAV antibodies when used as binding agent in binding assay format. Further provided is DNA encoding for these peptides.
In a first aspect of the present invention there is provided a peptide or peptide conjugate comprising one or more epitopes capable of evoking an immune response in animals producing antibodies which are neutralising to equine arteritis virus, characterised in that the epitopes are selected from those present in the amino acid sequence corresponding to amino acid 19 to 137 (SEQ ID No 3) of equine arteritis virus (EAV) GL protein; the peptide not being the GL protein.
Preferred peptides or peptide conjugates of the invention comprise the epitopes present in the amino acid sequence corresponding to amino acid 28 to 137 (SEQ ID No 4), more preferably 75 to 97 (SEQ ID No 5) and most preferably 85 to 97 (SEQ ID No 7) of EAV GL. Preferred peptides or peptide conjugates comprise the amino acid sequence corresponding to amino acid 75 to 97 or a sequence having at least 90% homology thereto; preferably comprising an amino acid sequence corresponding to a sequence at least 90% homologous to the sequence of amino acids 28 to 137 of equine arteritis virus GL protein (SEQ ID No 4), but including said 85 to 97, or more preferably the 75 to 97 sequence, or a sequence that has at least 90% homology thereto. Other desirable optional epitopes identified are at 33 to 44 and 53 to 64.
A second aspect of the present invention provides a peptide or peptide conjugate comprising one or more epitopes capable of evoking an immune response in animals that produces antibodies which are neutralising to equine arteritis virus, characterised in that the epitopes are selected from those present in the amino acid sequence corresponding to amino acid 19 to 137 of equine arteritis virus GL protein (SEQ ID No 3), for use as a diagnostic agent; such peptide or conjugate is particularly provided for use as a diagnostic agent for the detection of EAV. Such aspect of course includes equine arteritis virus GL protein as such for these uses. Peptides or conjugates comprising SEQ ID No 2 are preferred; GL protein being included for such use; but peptides or conjugates comprising an amino acid sequence corresponding to a sequence at least 90% homologous to the sequence of amino acids 19 to 137 of equine arteritis GL protein (SEQ ID No 3) or to SEQ ID No 4, while retaining the amino acids 75 to 97 (SEQ ID No 5 and most preferably retaining the amino acids 85 to 97 (SEQ ID No 7) of, or having at least 90% homology to, SEQ ID No 2 may be used.
In a third aspect of the present invention are provided compositions comprising isolated peptides or peptide conjugates as described above per se, including GL, particularly for use in evoking neutralising antibody responses, eg. for the purpose of prophylaxis or diagnosis. Typically such compositions will comprise a peptide or conjugate of the present invention together with a pharmaceutically acceptable carrier or a carrier suitable for use in binding studies respectively.
In a fourth aspect of the present invention there is provided recombinant DNA, or RNA derived therefrom, encoding for peptides or conjugates of the invention, and plasmids and cells transformed thereby comprising this DNA such that they are capable of expressing the peptides or conjugates. This DNA has sequences of SEQ ID Nos 3 to 7 and those indicated in Table 1 below, and may be incorporated into cells in the form of vectors such as plasmids or may be used as a xe2x80x98naked vaccinexe2x80x99 by way of chromosomal integration; both techniques being well understood by those skilled in the art.
In a fifth aspect of the present invention there is provided a method for testing for the presence of antibodies to equine arteritis virus comprising use of a peptide or peptide conjugate of the present invention, or GL protein, as a specific binding agent. Such test is preferably of ELISA format but may use the peptide or conjugate as immobilised binding agent or labelled secondary binding agent in a so called sandwich assay.
In binding assay where the peptide or peptide conjugate is immobilised this method may conveniently be carried out by use of commercially available assay plates onto which the peptide or conjugate is coated by suitable incubation in the known manner. For the purpose of assay a sample to be screened for EAV antibodies, eg. a serum sample, is typically incubated in contact with the plate, eg. in the wells, whereafter any EAV antibody present therein is identified by exposure to eg. an anti-horse IgA, IgG or IgM conjugated to a reporter group. Such reporter group may be in the form of a radiolabel, chemical label or a biological label. A typical biological label is an enzyme or cofactor, eg. biotin, and is detected by exposure to all the reactants necessary for a reporter reaction to occur dependent upon the presence of the reporter group. In the case of biotin the well may be exposed to streptavidin-peroxidase and then o-phenylenediamine dihydrochloride and the absorbance of the plate determined at 490 nm.
In a further example an immobilised anti-horse IgA, IgM or IgG antibody raised in another animal may be used to bind a specific class of horse antibody and then the immobilised horse antibody provided may be exposed to a solution containing labelled peptide or conjugate of the invention whereby presence of anti-EAV antibody is indicated by assay of the amount of label present. Other assay formats such as competitive assays using either bound and unbound peptide or conjugate will occur to those skilled in the art; these will include simple observation of agglutination between peptide or conjugate and the antibody in a simple dilution test.
In a further aspect of the present invention there are provided test kits for use in carrying out the assay of the invention characterised in that they comprise a peptide, peptide-conjugate or antibodies of the invention, together with optional agents and items necessary for performing such assays. Such agents and items may include other binding agents or colour forming agents such as labelled antibodies, eg. biotinylated anti-horse IgG, horseradish peroxidase, streptavidin-peroxidase conjugate and o-phenylenediamine dihydrochloride. It will be realised that the term peptide and peptide conjugate as used herein will encompass oligopeptides, polypeptides and proteins as long as they fulfil the criteria of the invention with regard to immunological activity and content of epitopic sequences. The term xe2x80x98conjugatexe2x80x99 designates conjugation to any physiologically acceptable entity.
The peptides, peptide conjugates and binding assays of the present invention will now be described by way of example only by reference to the following sequence listing, figures and examples.
Sequence Listing
SEQ ID No 1: is the DNA sequence equivalent to the entire EAV genome minus the first 18 bases and the polyA tail.
SEQ ID No 2: is the amino acid sequence corresponding to amino acids 1 to 137 of the EAV GL protein (including any signal sequence).
SEQ ID No 3: is the amino acid sequence corresponding to amino acids 19 to 137 of the EAV GL protein.
SEQ ID No 4: is the amino acid sequence corresponding to amino acids 28 to 137 of the EAV GL protein.
SEQ ID No 5: is the amino acid sequence corresponding to amino acids 75 to 97 of the EAV GL protein.
SEQ ID No 6: is the amino acid sequence that is fused with GST in Fp5.RsaI and used in the ELISA of Example 3.
SEQ ID No 7: is the amino acid sequence corresponding to the epitope at GL 85 to 97.